ENLARGED VESTIBULAR AQUEDUCTS (EVA) We ascertain families with multiple members with nonsyndromic EVA that is not associated with detectable SLC26A4 mutations or Pendred syndrome. Our hypothesis is that these families segregate recessive alleles at one or more other genetic loci that cause nonsyndromic EVA. We are using those families in a linkage-based exome sequencing strategy to identify other genetic causes of EVA. We used recombination breakpoint mapping to define a region of shared linkage overlap containing the SLC26A4 gene on chromosome 7 to search for occult (unidentified) mutations of SLC26A4 in families segregating nonsyndromic EVA with only one detectable mutant allele of SLC26A4. Our hypothesis is that these families segregate a second, unidentified, mutation of SLC26A4. We are using massively parallel sequencing to sequence the entire region of shared overlap to identify occult mutations. We previously generated a doxycycline-inducible Slc26a4-expression mouse line. This transgenic mouse line allows us to manipulate Slc26a4 expression (on an Slc26a4-knockout background) by the administration of doxycycline in drinking water. We manipulated doxycycline administration to generate mice in which there is significant residual hearing and isolated EVA at the age of one month. Longitudinal analysis of these mice revealed large fluctuations of hearing from 1-3 months of age, followed by progressive hearing loss from 9-12 months of age. The hearing loss is associated with reduction of the endocochlear potential, pathology of the intermediate layer of the stria vascularis, which generates the endocochlear potential, and evidence of oxidative stress and damage (ref. 1). The pattern of hearing loss remarkably resembles that observed in many human patients with EVA and validates the potential of this animal model to further explore the pathophysiology and potential therapeutic interventions to prevent hearing loss fluctuation and progression. We are currently testing the ability of Slc26a4 expression to stabilize hearing and prevent fluctuations in mature ears of our mouse model. We are completing an analysis of the functional and pathologic changes in the ears of our model mice from 6-12 months of age, when they have steadily downward progression of hearing. We are measuring hearing every 2-3 days in the model mice to more precisely characterize the temporal and pathologic characteristics associated with fluctuating hearing loss between the ages of 1 and 3 months. TMC GENES We previously generated and reported mice with knockout (null) alleles of Tmc1 and Tmc2. We had shown that Tmc1 and Tmc2 are functionally redundant and required for mechanotransduction in the stereocilia of postnatal cochlear and vestibular sensory hair cells (reviewed in refs. 2 and 3). The results suggested that TMC1 and TMC2 may comprise the hair cell mechanoelectrical transduction channel, or are intimately involved in its development and/or function. We are continuing to test this hypothesis by localization of TMC1 and TMC2 proteins in hair cells. We also collaborated with Dr. Jeffrey Holt to characterize the whole-cell and single-channel mechanoelectrical transduction currents in mice segregating mutant alleles of Tmc1 and Tmc2. The results show distinctly different hair cell single-channel transduction current properties (ion permeability and conductance) associated with the expression or presence of TMC1 in comparison to TMC2 in comparison to a mutated form of TMC1. The data are consistent with the hypothesis that TMC1 and TMC2 are components of the mechanotransduction channel. This study was published in Neuron (ref. 4). We generated knockout mice for Tmc6 and Tmc8 to better understand the function(s) of Tmc genes and proteins. Mutations in human TMC6 or TMC8 genes cause epidermodysplasia verruciformis, a recessive disease resulting in chronic cutaneous HPV infections (papillomas or warts) with increased susceptibility to non-melanoma skin cancers. We have done extensive RNA expression analyses to show that Tmc6 and Tmc8 are primarily expressed in lymphoid cells and tissues and lung and skin, and primarily during development. The homozygous knockout mice have no obvious phenotypic abnormalities, so we are collaborating with Dr. Paul Lambert to determine if these mice have alterations in their susceptibility or response to papillomavirus infection and progression to non-melanoma skin cancers. DFNA34 HEARING LOSS We mapped a novel nonsyndromic hearing loss locus, DFNA34, in a single large family. We used recombinations to define a critical map interval in which the gene and mutation must be located. We identified a likely mutation in a gene in which other mutations cause hearing loss associated with autoinflammatory disease. In order to confirm this mutation as causative, we used massively parallel sequencing as well as conventional Sanger dideoxy sequencing to rule out mutations in any of the other genes in the critical map interval. We detect expression of the candidate gene in the inner ear. We are collaborating with Drs. Daniel Kastner and Raphaela Goldbach-Mansky to study the patients for evidence of cochlear and systemic auto-inflammation on magnetic resonance imaging studies at the NIH Clinical Center. We have detected sub-clinical evidence of systemic and cochlear auto-inflammation, providing conclusive proof of the pathogenic nature of the mutation we have detected. COLLABORATIVE PROJECTS We collaborated with Dr. Thomas Friedman and others in a project that identified mutations of the TBC1D24 gene as the cause of non syndromic recessive deafness DFNB86 and to show that the TBC1D24 gene is expressed in spiral ganglion neurons (ref. 5). We collaborated with Drs. Kelly King and Carmen Brewer and others to detect hearing loss and the associated pathology in a mouse model of Niemann-Pick Type C disease (ref. 6).